Beard et al. Parasites Vectors (2021) 14:60 https://doi.org/10.1186/s13071-020-04565-6 Parasites & Vectors RESEARCH Open Access Morphological identifcation of ticks and molecular detection of tick-borne pathogens from bare-nosed wombats (Vombatus ursinus) Danielle Beard1, Hayley J. Stannard2 and Julie M. Old1* Abstract Background: Ticks are obligate haematophagous ectoparasites of vertebrate hosts and transmit the widest range of pathogenic organisms of any arthropod vector. Seven tick species are known to feed on bare-nosed wombats (Vom- batus ursinus), in addition to the highly prevalent Sarcoptes scabiei mite which causes fatal sarcoptic mange in most bare-nosed wombat populations. Little is known about the pathogens carried by most wombat ticks or how they may impact wombats and wombat handlers. Methods: Wombat ticks were sourced from wildlife hospitals and sanctuaries across Australia and identifed to spe- cies level using taxonomic keys. Genomic DNA was extracted from a subsample, and following the amplifcation of the bacterial 16S rRNA gene V3–V4 hypervariable region, next-generation sequencing (NGS) on the Illumina MiSeq platform was used to assess the microbial composition. Results: A total of 447 tick specimens were collected from 47 bare-nosed wombats between January 2019 and Janu- ary 2020. Five species of ticks were identifed comprising wombat tick Bothriocroton auruginans (n 420), wallaby tick Haemaphysalis bancrofti (n 8), bush tick Haemaphysalis longicornis (n 3), common marsupial tick= Ixodes tasmani (n 12), and Australian paralysis= tick Ixodes holocyclus (n 4). Tick infestations= ranged from one to 73 ticks per wombat. The= wombat tick was the most prevalent tick species comprising= 94% of the total number of samples and was pre- sent on 97.9% (46/47) of wombat hosts. NGS results revealed the 16S rRNA gene diversity profle was predominantly Proteobacteria (55.1%) followed by Firmicutes (21.9%) and Actinobacteria (18.4%). A species of Coxiella sharing closest sequence identity to Coxiella burnetii (99.07%), was detected in 72% of B. auruginans and a Rickettsiella endosymbiont dominated the bacterial profle for I. tasmani. Conclusions: A new host record for H. longicornis is the bare-nosed wombat. One adult male and two engorged adult female specimens were found on an adult male wombat from Coolagolite in New South Wales, and more specimens should be collected to confrm this host record. The most prevalent tick found on bare-nosed wombats was B. auruginans, confrming previous records. Analysis of alpha-diversity showed high variability across both sample locations and instars, similar to previous studies. The detection of various Proteobacteria in this study highlights the high bacterial diversity in native Australian ticks. Keywords: Wombat, Tick, Microbiome, Marsupial, 16S ribosomal RNA gene, Next-generation sequencing, Bacteria *Correspondence: [email protected] 1 School of Science, Western Sydney University, Penrith, New South Wales, Australia Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Beard et al. Parasites Vectors (2021) 14:60 Page 2 of 18 Background with native tick species, and healthy wombats regularly Ticks (Acari: Ixodidae) are obligate ectoparasitic arach- carry large burdens of ticks which would otherwise afect nids that are classifed into three families: Ixodidae (hard humans and domestic animals [20]. However, wombats ticks), Argasidae (soft ticks), and Nuttalliellidae. Each of afected by sarcoptic mange, orphaned or injured wom- the three families have evolved unique biological, physi- bats released from captivity and wombats raised in a ological and ecological disparities which have resulted comparatively parasite-free captive environment before in diferent abilities and capacities to transmit patho- release are likely at an increased risk of contracting tick- gens [1]. However, ticks can transmit the widest range of borne diseases. Managing wild species in captivity may pathogens of any arthropod vector and are the primary induce stress, impair immunity and expose hosts to novel cause of vector-borne diseases in livestock and domestic parasites to which the immune system is naïve [21]. Pop- animals [2]. Ixodids transmit the widest number of path- ulation density is also often atypical in captivity, which ogens worldwide and are responsible for the majority of may result in higher than usual parasite burdens. Addi- tick-borne infections [3]. tionally, the use of anti-parasitic medications on captive In addition to pathogens, the tick microbiome com- animals afects both host-parasite relationships and indi- prises a community of commensal and symbiotic obli- viduals, as the latter are at an increased risk of disease gate endosymbionts which make up the majority of the once released, having not developed acquired immunity tick microbiome and reside both inside and outside the [22]. body of ticks [4]. Te efect of these organisms has been Seven species of ticks have previously been recorded somewhat neglected in studies, but may present various feeding on bare-nosed wombats including the wom- detrimental, neutral, or benefcial efects to their tick bat tick Bothriocroton auruginans [23][23], wallaby tick hosts, and also contribute to driving the transmission of Haemaphysalis bancrofti [24], Australian paralysis tick tick-borne pathogens [5]. Non-pathogenic microorgan- Ixodes holocyclus [25], Tasmanian paralysis tick Ixodes isms are typically transovarially transmitted [6] and may cornuatus [26], Ixodes phascolomyis [27], common mar- impact tick growth, reproduction, ftness, nutritive adap- supial tick Ixodes tasmani and Ixodes victoriensis [28] tation and defence against environmental stresses [7, 8]. [28]. Te relationship between S. scabiei and other Te functional roles of tick microorganisms and their known wombat ectoparasites, their pathogens, ability to relationships may provide further insights into the path- co-infect hosts, and their overall impact on wombat hosts ogenicity and evolution of tick pathogens. For example, have not yet been investigated. Tere is also very little it has become increasingly clear since the advancement known about the life cycles of wombat ectoparasites and of molecular barcoding techniques that many species of their level of host specifcity. Coxiella burnetii has been Rickettsia, Francisella, and Coxiella, which are generally found in B. auruginans collected from bare-nosed wom- considered pathogens of medical and veterinary impor- bats, as well as a Rickettsia species closely related to Rick- tance, have evolved as non-pathogenic endosymbionts of ettsia massiliae, which causes human disease [29]. Tese ticks [9]. are the only pathogens that have been detected in ticks While tick-borne bacteria have been relatively well taken from wombat hosts, and were identifed using spe- studied in the northern hemisphere, very little is known cifc targeted methods. about the presence or diversity of bacteria in Australian Te development of next-generation sequencing (NGS) ticks [10]. Te microbiome and pathogenicity of Austral- technologies has enabled the microbial communities ian ticks are unique when compared to other species, and of ticks to be explored in a fast and cost-efcient man- so is the response to ticks and tick-borne pathogens from ner [15]; however, very little is known about the micro- native vertebrate hosts [11]. Recently, unique Australian biome of native Australian ticks [10] and no studies species of Anaplasma, Ehrlichia and Neoehrlichia [12, have focused on wombat ticks or tick-borne pathogens. 13] and the frst native Borrelia species were character- Bare-nosed wombats are already signifcantly afected ised in native ticks [14]. Other novel microbial species by a known ectoparasite, so it would be benefcial to have also been reported in Australian ticks [12, 15, 16]; understand the other parasitic and pathogenic threats however, the focus has largely been on ticks of human, that wombats may need to overcome simultaneous to domestic animal and livestock importance, and few stud- or following the treatment of sarcoptic mange. It is also ies have surveyed ticks associated with wildlife [17, 18]. important to identify potential zoonotic threats to wom- Bare-nosed wombat (Vombatus ursinus) populations bat handlers and domestic animals that may come into are signifcantly impacted by the ectoparasite Sarcoptes contact with wombats or their burrows. Tis study